In the mammalian testis, spermatogenesis takes place in the epithelium of the seminiferous tubule, the functional unit that produces millions of spermatozoa daily. The blood-testis barrier (BTB), located near the basement membrane (BM) of the seminiferous epithelium, confers gate-keeper and the fence function, which restricts the paracellular and transcellular transport of harmful substances such as toxicants across the barrier, respectively, to maintain spermatogenesis. The BTB also physically divides the seminiferous epithelium into a basal and an adluminal compartment; thus, meiosis I and II, and post-meiotic spermatid development take place in a specialized microenvironment (i.e., the adluminal compartment) behind the BTB. The BTB is one of the tightest blood-tissue barriers in mammals since cell junctions at the BTB are reinforced by bundles of actin filaments that lie perpendicular to apposing Sertoli cell plasma membranes, which are only found in the testis. However, the BTB is a dynamic structure, undergoing extensive restructuring at stage VIII of the epithelial cycle so that preleptotene spermatocytes can cross the BTB and enter the adluminal compartment for further development. It is now well established that a functional BTB is crucial for spermatogenesis. Studies completed in the past grant period have shown that BTB function is regulated by biologically active peptides released (i) at the apical ectoplasmic specialization (apical ES, a testis-specific adherens junction at the Sertoli-spermatid interface), and (ii) at th basement membrane (BM) mediated by a functional axis designated the apical ES-BTB-BM axis. In short, biologically active fragments of laminin and collagen chains released at the apical ES and the BM, respectively, act as autocrine factors to regulate BTB function in this axis. Herein, we will continue this important research to understand the underlying molecular mechanism(s) that regulates BTB function. First, we will unravel the mechanism(s) by which these fragments regulate Sertoli cell adhesion and BTB restructuring via their effects on the F-actin network at the BTB, such as actin binding and regulatory proteins. We will also examine the involvement of endosome-mediated protein trafficking and small regulatory RNAs in these events using state-of-the-art techniques and approaches. Second, we will identify the biological significance of biomolecules, such as cytokines, steroids and matrix metalloproteases, in regulating the stage-specific production of these bioactive peptides during the epithelial cycle of spermatogenesis. These findings will have a significant impact in understanding the biology of BTB in spermatogenesis. More important, these findings will shed new light on the development of novel reversible non-hormonal contraceptives for men. For instance, peptides derived from the apical ES and BM can be developed into contraceptives to perturb BTB function and impair spermatogenesis. Additionally, these findings can lead to the development of new therapeutic approaches to interfere with toxicant-induced male reproductive dysfunction, which is one of the leading causes of the decline in male fertility. For instance, the environmental toxicant cadmium mediates its effects at the BTB to induce male reproductive dysfunction; these findings provide crucial information in the design and synthesis of small molecules that either reverse or block cadmium-induced BTB disruption that leads to male infertility.